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FMDV全ORF编码基因的克隆及其腺病毒穿梭质粒的构建
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张兴旺1，2，3，王勤2，柳纪省1，李志勇1，王辉1，殷相平1，谢庆阁1
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（１.中国农业科学院 兰州兽医研究所，甘肃 兰州730046； ２.兰州大学 生命科学学院，
甘肃 兰州730000； 3.甘肃省人民医院检验中心，甘肃 兰州730000)
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摘要：
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为研制FMD复制缺陷型腺病毒活载体疫苗，通过RTPCR方法获得了O型口蹄疫病毒（FMDV）全开放阅读框（ORF）基因片段，将其克隆到pMD18T载体后测序，再将ORF编码基因定向克隆入腺病毒穿梭载体pAdTrackCMV中，构建了重组腺病毒穿梭质粒。经PCR、酶切及测序鉴定，所克隆的ORF编码基因序列与原始强毒株Akesu/58的ORF编码基因比较，L、P1、P2、P3基因的核苷酸同源性分别为98.8%、97.9%、99.0%和97.6%，PCR、酶切鉴定重组腺病毒穿梭质粒均获得了长约6.9kb的目的基因片段。表明，成功获得了含有O型FMDV全ORF编码基因的阳性克隆，并成功构建了含有完整FMDV开放阅读框架的编码基因表达盒的重组腺病毒穿梭质粒。 |
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关键词：
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口蹄疫病毒； 全开放阅读框； 克隆； 穿梭载体 |
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中图分类号：S 852.659.6：Q 785 文献标识码：
A 文章编号：1673-4696(2006)02-0093-05 |
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Cloning of whole ORF coding genes of FMDV and construction of
recombinant adenoviral pAdTrackCMV |
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ZHANG Xingwang1,2,3, WANG Qin2,LIU
Jixing1, LI Zhiyong1，
WANG Hui1, YIN Xiangping1，XIE Qingge1 |
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(1.Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Lanzhou 730046, China ;
2. School of Life Science, Lanzhou University, Lanzhou 730000, China;
3. Center of Clinical Laboratory, People’s Hospital of Gansu Province, Lanzhou 730000, China) |
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Abstract: In order to develop a recombinant adenoviral pAdTrackCMV vaccine, genes of a whole ORF coding genes of footandmouth disease virus(FMDV) were amplified by RTPCR and inserted subsequently into a pMD18T vector and sequenced. The gene was cloned into the vector pAdTrackCMV to construct the recombinant adenoviral pAdTractCMV/ORF. The recombinant pAdTrack/ORF was identified by PCR，digestion with restriction endonucleases and sequencing. The sequencing results showed that the amplified L, P1, P2 and P3 gene’s nucleotide sequences shared 98.8% ,97.9% ,99.0% and 97.6% homology with that of Akesu/58, respectively. PCR amplification and digestion with XbaⅠ+NotⅠ confirmed that the recombinant adenoviral pAdTrackCMV contained the target genes of 6.9kb. The results showed that the recombinant pAdTrack/ORF containing the whole ORF gene of O type FMDV was constructed successfully. |
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Key words: footandmouth disease virus; open reading frame; cloning; pShuttle vector |
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